A time-resolved, multi-symbol molecular recorder via sequential genome editing.
A time-resolved, multi-symbol molecular recorder via sequential genome editing.
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DOI：
10.1038/s41586-022-04922-8
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发表时间：
2022-08
期刊：
影响因子：
64.8
通讯作者：
Shendure, Jay
中科院分区：
文献类型：
--
作者：
Choi, Junhong;Chen, Wei;Minkina, Anna;Chardon, Florence M.;Suiter, Chase C.;Regalado, Samuel G.;Domcke, Silvia;Hamazaki, Nobuhiko;Lee, Choli;Martin, Beth;Daza, Riza M.;Shendure, Jay
DNA自然非常适合用作体内分子记录的数字介质。然而，当代的基于DNA的存储器设备在可以同时记录的不同“符号”的数量方面和/或通过无法捕获事件发生的顺序而受到限制。在这里，我们描述DNA打字机，一个通用的系统，在体内分子记录，克服了这些和其他限制。对于DNA打字机，空白记录介质（“DNA磁带”）由部分CRISPR-Cas9靶位点的串联阵列组成，其中除了第一个位点之外的所有位点在其5 '端被截短，因此是无活性的。短插入编辑用作记录介导编辑的主要编辑向导RNA的身份的符号，同时还将“类型向导”的位置沿着DNA带移动一个单位，即顺序基因组编辑。在DNA打字机的概念证明中，我们演示了数千个符号，复杂事件历史和短文本消息的记录和解码;评估了数十个正交磁带的性能;并构建了可能能够记录多达20个串行事件的“长磁带”。最后，我们利用DNA Typewriter结合单细胞RNA-seq重建了3，257个细胞的单系谱系，并发现对多拷贝DNA带的顺序编辑的泊松样积累可以在至少20代和25天的体外克隆扩增中保持。一种DNA记忆设备，DNA打字机，使用顺序的素数编辑来记录多个细胞事件的顺序。
DNA is naturally well suited to serve as a digital medium for in vivo molecular recording. However, contemporary DNA-based memory devices are constrained in terms of the number of distinct ‘symbols’ that can be concurrently recorded and/or by a failure to capture the order in which events occur. Here we describe DNA Typewriter, a general system for in vivo molecular recording that overcomes these and other limitations. For DNA Typewriter, the blank recording medium (‘DNA Tape’) consists of a tandem array of partial CRISPR–Cas9 target sites, with all but the first site truncated at their 5′ ends and therefore inactive. Short insertional edits serve as symbols that record the identity of the prime editing guide RNA mediating the edit while also shifting the position of the ‘type guide’ by one unit along the DNA Tape, that is, sequential genome editing. In this proof of concept of DNA Typewriter, we demonstrate recording and decoding of thousands of symbols, complex event histories and short text messages; evaluate the performance of dozens of orthogonal tapes; and construct ‘long tape’ potentially capable of recording as many as 20 serial events. Finally, we leverage DNA Typewriter in conjunction with single-cell RNA-seq to reconstruct a monophyletic lineage of 3,257 cells and find that the Poisson-like accumulation of sequential edits to multicopy DNA tape can be maintained across at least 20 generations and 25 days of in vitro clonal expansion. A DNA memory device, DNA Typewriter, uses sequential prime editing to record the order of multiple cellular events.
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DOI：
10.1126/science.1256272
发表时间：
2014-11-14
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Farzadfard F;Lu TK
通讯作者：
Lu TK
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
16
作者：
Farzadfard, Fahim;Gharaei, Nava;Lu, Timothy K.
通讯作者：
Lu, Timothy K.
DOI：
10.1126/science.aaf7907
发表时间：
2016-07-29
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
McKenna A;Findlay GM;Gagnon JA;Horwitz MS;Schier AF;Shendure J
通讯作者：
Shendure J
影响因子：
14.8
作者：
Loveless TB;Grotts JH;Schechter MW;Forouzmand E;Carlson CK;Agahi BS;Liang G;Ficht M;Liu B;Xie X;Liu CC
通讯作者：
Liu CC